Project Summary The long term goals of this research program is to illuminate the mechanistic principles that describe small heat-shock proteins (sHSP) chaperone activity, reveal the sequence and structural elements underlying their oligomer dynamics and polydispersity, and define their physiological roles in the context of the proteostasis network. The focus has been historically on lens ?-crystallins which are hypothesized to buffer protein aggregation of damaged lens proteins in the absence of turnover thereby delaying the onset of cataract. A thermodynamic model of the interaction of sHSP with client proteins and an integrated structural biology approach has long underpinned our research program. With the recent spectacular progress in the genetics of zebrafish (D. rerio), this program has transitioned to the phase of testing mechanistic models in a living animal. Premised on a detailed mechanistic model and grounded in discoveries in the previous funding period, this renewal will test two hypotheses describing the physiological roles of ?-crystallins. Aims 1 and 2 will use zebrafish lines genetically engineered with compromised oxidative response, reduced chaperone capacity or expressing truncated crystallins to experimentally test the long-standing notion in the field that ?-crsyatllins bind and sequester aggregation-prone proteins in vivo. Aim 3 will follow up on our recent discovery that ?B-crystallin is critical for survival of zebrafish under stress. The mechanistic underpinning of this finding is a direct link to glucocorticoid-activated signaling pathways and is mediated by interactions with filament proteins. The design of these aims will leverage our protein engineering efforts which have yielded variants with designed chaperone activity, including phosphorylation mimics of ?B- crystallin. In addition to revealing the physiological roles of sHSP, the research plan will have direct impact on current efforts for therapeutic interventions to delay or treat cataract. Zebrafish models generated in the context of this application will provide a valuable tool for high throughput screening.